Characterization of a human 5‐hydroxytryptamine3 receptor type A (h5‐HT3R‐AS) subunit stably expressed in HEK 293 cells

1. A cloned cDNA encoding a human 5-hydroxytryptamine receptor type A subunit (h5-HT₃R-A(s)) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected. 2. Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (B(max) = 4.49 ± 0.46 pmol mg^-1 protein) of sites that bound the radiolabelled 5-HT₃ receptor antagonist, [³H]-granisetron with high affinity (pK(D) = 8.87 ± 0.08). Kinetic studies (at 37°C) revealed rapid association (κ₊₁ = 4.76 ± 0.3 x 10⁸ M^-1 min^-1) and dissociation (κ_-1 = 0.21 ± 0.003 min^-1) of the radioligand. 3. Selective and non-selective 5-HT₃ receptor ligands competed for [³H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide >> phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT₃ receptors endogenous to the human CNS. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 μM) evoked transient inward current responses that reversed in sign at a potential of -1.0 ± 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5. The construction of a stable cell line expressing a high density of recombinant human 5-HT₃ receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT₃ receptor pharmacology.